Cell Electrofusion Visualized with Fluorescence Microscopy

Trontelj, Katja; Ušaj, Marko; Miklavčič, Damijan

doi:10.3791/1991

Cited by 6 publications

(4 citation statements)

References 10 publications

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“…However, treatment with PEG showed toxic effects on cells and therefore we continued only with Syncytin1 overexpression for further experiments. We then tested electrofusion – the use of high voltage electrical pulses to induce fusions of cells in close proximity 60 – on an admixture of the cells expressing either halve. For this aim, different voltages were tested: 300V 500 µs one pulse vs 700V 15 µs three pulses.…”

Section: Resultsmentioning

confidence: 99%

Synthetic Circuits Based on Split Cas9 to Detect Cellular Events

Przybyszewska-Podstawka

Czapiński

Kałafut

et al. 2023

Preprint

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Synthetic biology involves the generation of logic circuits to create or control biological functions and behaviours by engineering interconnected genetic elements such as promoters, repressors, and transcriptional activators. CRISPR discovery, and its adaptations to mammalian cells, has made it the tool of choice in molecular biology and revolutionized genome engineering in biomedical sciences. Here, we describe an adaptation of a split Cas9 to generate synthetic logic gates to sense biological events. As proof-of-concept, the complementing halves of split Cas9 were placed under different promoters, one unique to cancer cells of epithelial origin (phCEA) and one universal promoter (pCMV). We used self-assembling inteins to reunite the halves when co-expressed. Only cancer cells with epithelial origin expressed both halves and activated a reporter becoming green fluorescent. We then investigated whether we could apply this system to the detection of biological processes such as epithelial to mesenchymal transition (EMT). We designed another logic gate where one halve is expressed only by cancer cells of epithelial origin, while the other is activated during EMT - under the control of TWIST1. Indeed, cells undergoing EMT were detected by the activation of the reporter. Finally, the split-Cas9 logic gate was applied as a sensor to detect cell-cell fusion events in multiple cell lines. Each cell type expressed only one halve of split Cas9, and only induction of fusion resulted in the appearance of multinucleated syncytia and the expression of the reporter system. The simplicity and flexibility of the split Cas9 system reported here can be integrated to many other cellular processes, not only as a sensor but as an actuator.

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Section: Resultsmentioning

confidence: 99%

Synthetic Circuits Based on Split Cas9 to Detect Cellular Events

Przybyszewska-Podstawka

Czapiński

Kałafut

et al. 2023

Preprint

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“…Subsequently, other research groups performed similar electrofusion studies using mouse melanoma cells (B16-F1) and mouse embryonic cells. The results showed that the optimal electric field intensity values were 1200 V/cm [35] and 800 V/cm [36] , respectively. According to the previous studies, there is a slight difference in optimal electric field intensity in different cells.…”

Section: Cell Fusionmentioning

confidence: 99%

“…The Zimmermann group suggested that the pulse duration should not exceed 100 μs, otherwise it would cause irreversible electroporation, resulting in cell lysis. In recent years, microsecond (μs)-level pulses have been widely applied in cell electrofusion experiments [35,36] . Nevertheless, the microsecond-level pulse duration is still relatively long and will lead to a large electroporation radius, causing serious irreversible damage to the cell membrane and ultimately resulting in a significant decrease in cell fusion yield.…”

Section: Cell Fusionmentioning

confidence: 99%

Advances in hybridoma preparation using electrofusion technology

Kou

Shen

wang

et al. 2022

Preprint

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As a rapidly developing cell engineering technique, cell electrofusion has been increasingly applied in the field of hybridoma preparation in recent years. However, electrofusion is a certain degree of difficulty to completely replace the polyethylene glycol-mediated cell fusion. The key elements limiting electrofusion in the field of hybridoma preparation are practical complicated. This review summarizes the state of art of cell electrofusion in hybridoma preparation based on recent published literatures, mainly focusing on electrofusion instruments and their components, process control, cell treatment, and process characterization. The review provides new information and insightful commentary, critically important to the promotion of further electrofusion development in the field of hybridoma preparation.

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“…For treatments using hypoosmolar buffer, a wash step using isoosmolar buffer prior to PEG treatment was used. Isoosomolar potassium phosphate buffer (10 mM KH2PO4, 10 mM K2HPO4, 1mM MgCl2, and 250 mM sucrose, in dH2O) and hypoosmolar potassium phosphate buffer (10 mM KH2PO4, 10 mM K2HPO4, 1mM MgCl2, 75 mM sucrose, in dH2O) were prepared as previously described [22], filter-sterilized, and stored at 4°C before use. Every treatment was replicated four times, with 1 to 3 wells each time.…”

Section: Homokaryon Productionmentioning

confidence: 99%

Production of interspecies somatic/pluripotent heterokaryons using polyethylene glycol (PEG) and selection by imaging flow cytometry for the study of nuclear reprogramming

et al. 2020

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Fusion of somatic cells to pluripotent cells such as mouse embryonic stem (ES) cells induces reprogramming of the somatic nucleus, and can be used to study the effect of trans-acting factors from the pluripotent cell on the somatic nucleus. Moreover, fusion of cells from different species permits the identification of the transcriptome of each cell, so the gene expression changes can be monitored. However, fusion only happens in a small proportion of the cells exposed to fusogenic conditions, hence the need for a protocol that produces high fusion rate with minimal cell damage, coupled with a method capable of identifying and selecting fusion events from the bulk of the cells. Polyethylene glycol (PEG) is a polymer of repeated ethylene oxide units known to induce cell fusion within a certain range of molecular weight. Here, we describe a method to induce formation of bi-species heterokaryons from adherent mammalian cells, which can then be specifically labeled and selected using live cell immunostaining and a combination of imaging and traditional flow cytometry. First, we tested several PEG-based fusion conditions to optimize a protocol to consistently produce both mouse NIH/3T3 fibroblast and primary bovine fetal fibroblast (bFF) homokaryons. Initially, we obtained 7.28% of NIH/3T3 homokaryons when using 50% PEG 1500. Addition of 10% of DMSO to the PEG solution increased the percentage of NIH/3T3 homokaryons to 11.71%. In bFFs, treatment with 50% PEG 1500 plus 10% DMSO produced 11.05% of homokaryons. We then produced interspecies heterokaryons by fusing mouse embryonic stem (mES) cells to bFFs. To identify bi-species fusion products, heterokaryons were labeled using indirect immunostaining in live cells and selected using imaging (Amnis ImageStream Mark II) and traditional (BD FACSAria I) flow cytometry. Heterokaryons selected with this method produced ES cell-like colonies when placed back in culture. The method described here can also be combined with downstream applications such as nucleic acid isolation for RT-PCR and RNA-seq, and used as a tool to study cellular processes in which the effect of trans-acting factors is relevant, such as in nuclear reprogramming.

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Cell Electrofusion Visualized with Fluorescence Microscopy

Cited by 6 publications

References 10 publications

Synthetic Circuits Based on Split Cas9 to Detect Cellular Events

Synthetic Circuits Based on Split Cas9 to Detect Cellular Events

Advances in hybridoma preparation using electrofusion technology

Production of interspecies somatic/pluripotent heterokaryons using polyethylene glycol (PEG) and selection by imaging flow cytometry for the study of nuclear reprogramming

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